PHOTOLABELING OF ACETYLCHOLINE BINDING PROTEIN BY NICOTINOIDS & NEONICOTINOIDS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 烟碱型乙酰胆碱受体（nAChR）是一种典型的激动剂门控离子通道，负责快速兴奋性神经传递。nAChR激动剂包括许多毒物、潜在的治疗剂和重要的烟碱类杀虫剂。了解受体-激动剂相互作用的最初尝试涉及定点或嵌合诱变，估计特定区域或氨基酸对药理学反应（电生理学或配体结合）的作用。高分辨率X射线晶体学的结构生物学方法揭示了药物结合状态下功能氨基酸的几何结构和配体相互作用后蛋白质的构象重排。或者，掺入非天然氨基酸或光亲和标记提供了配体结合环境的识别特性的直接和生理学相关的定义。烟碱和类烟碱激动剂的结合位点相互作用的特征在于比较结构和化学生物学方法使用软体动物乙酰胆碱结合蛋白（AChBP），这是一个合适的结构替代物的nAChR的胞外配体结合域。5-叠氮基-6-氯吡啶-3-基烟碱光亲和探针在研究激动剂-nAChR相互作用中发挥了重要作用。原则上，这些探针结合到特定位点，然后通过光照射产生的反应性氮烯中间体与靶蛋白共价反应。在光亲和标记的AChBP，叠氮氯吡啶基光探针衍生化之间的主要或（+）-面亚基和环E M116的合作或（-）-面亚基的环C Y195和环E M116之间的界面，建立一个结合的配体的位置和构象相同的晶体结构中观察到的。有趣的是，这种类型的烟碱光亲和配体专门标记鸡<$4 <$2 nAChR亚型的<$4亚基，其中<$4亚基上的环C Y225和<$2亚基上的环E F137分别与AChBP的Y195和M116在空间上等同。这些观察结果可能是合理的氨基酸和光活化的探针分子之间的反应性的差异。因此，本研究设计了一种蛋氨酸和酪氨酸扫描方法，主要是对环E域涉及17 AChBP突变体的光反应性的5-叠氮基-6-氯吡啶-3-基烟酸光探针，最终映射的特定网站进行光衍生化的质谱分析和精确的位置和构象的结合配体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The nicotinic acetylcholine (ACh) receptor (nAChR) is the prototypical agonist-gated ion channel responsible for rapid excitatory neurotransmission. The nAChR agonists include many toxicants, potential therapeutic agents, and the important neonicotinoid insecticides. Initial attempts to understand receptor-agonist interactions involved site-directed or chimeric mutagenesis, estimating the role of specific region(s) or amino acid(s) on the pharmacological response (electrophysiological or ligand binding). The structural biology approach of high-resolution X-ray crystallography reveals geometries of functional amino acids in the drug-bound state and conformational rearrangement of the protein upon ligand interaction. Alternatively, incorporation of an unnatural amino acid or photoaffinity labeling provides a direct and physiologically-relevant definition of the recognition properties of the ligand binding environment. Binding site interactions of nicotinoid and neonicotinoid agonists have been characterized by comparative structural and chemical biology approaches using mollusk ACh binding protein (AChBP), which is a suitable structural surrogate of the extracellular ligand-binding domain of the nAChR. 5-Azido-6-chloropyridin-3-yl nicotinic photoaffinity probes have played an important role in studying agonist-nAChR interactions. In principle, these probes bind to the specific site and then the reactive nitrene intermediate, generated by photoirradiation, reacts covalently with the target protein. On photoaffinity labeling of Aplysia californica AChBP, the azidochloropyridinyl photoprobes derivatized at only one position at the interface between loop C Y195 on the principal or (+)-face subunit and loop E M116 on the partnering or (-)-face subunit, establishing a bound ligand position and conformation identical to those observed in crystal structures. Interestingly, a nicotinic photoaffinity ligand of this type exclusively labeled the ¿4 subunit of the chick ¿4¿2 nAChR subtype, in which loop C Y225 on the ¿4 subunit and loop E F137 on the ¿2 subunit are spatially equivalent to Y195 and M116, respectively, of AChBP. These observations may be rationalized by the difference in the reactivity between amino acids and the photoactivated probe molecule. Therefore, the present investigation designs a methionine- and tyrosine-scanning approach principally on the loop E domain involving 17 AChBP mutants regarding photoreactivity of the 5-azido-6-chloropyridin-3-yl nicotinic photoprobes, ultimately mapping the specific site undergoing photoderivatization by mass spectrometry analyses and precise position and conformation of the bound ligand.